Transcriptional control of the H-NS antagonists LeuO and RcsB-BglJ in Escherichia coli

The bacterial nucleoid-associated protein (NAP) H-NS is involved in the organization and compaction of the bacterial chromatin and acts as a global respressor, mainly of genes that have been acquired by horizontal gene transfer and that are related to stress responses and pathogenicity. Binding of H-NS to the DNA and formation of a nucleoprotein complex at promoter regions leads to repression. This repressor effect of H-NS can be antagonized by gene-specific transcription factors (H-NS antagonists) that activate transcription of H NS-repressed genes by competing with H-NS for binding or by disturbing formation of the nucleoprotein complex. Two examples of such H NS antagonists are the LysR-type transcription factor LeuO and the FixJ/NarL-type transcription factor heterodimer RcsB-BglJ. LeuO is a pleiotropic regulator of stress responses and virulence determinants. RcsB-BglJ activates transcription of the H NS-repressed bgl (aryl-β,D-glucoside) operon. In this work, novel targets of RcsB-BglJ were identified in Escherichia coli by microarray analyses. The results suggest that heterodimerization of RcsB and BglJ is essential for regulation. Further, in addition to genes related to unknown or predicted function in the membrane the leuO gene was identified as a target gene. Detailed analysis of transcriptional regulation of leuO demonstrated that RcsB-BglJ strongly activates transcription of leuO by binding proximal to a newly mapped leuO promoter. Thus RcsB-BglJ antagonizes repression of leuO by H-NS and the H-NS-like protein StpA. Additional data presented here show that LeuO negatively autoregulates its own expression and inhibits activation of leuO by RcsB-BglJ. Regulation of leuO by RcsB-BglJ and autoregulation by LeuO, as shown here, as well as activation of bglJ by LeuO, as published previously, indicates a feedback control mechanism of two global transcriptional regulators and H-NS antagonists.This feedback regulation may ensure turn on of their expression in response to specific environmental signals. Screens to search for novel regulators or upstream signals were performed by transposon mutagenesis and by using a genomic expression library. These screens indicate that additional factors may be involved in the regulation of this leuO-bglJ feedback loop

Spindle-E acts antivirally against alphaviruses in mosquito cells

Mosquitoes transmit several human- and animal-pathogenic alphaviruses (Togaviridae family). In alphavirus-infected mosquito cells two different types of virus-specific small RNAs are produced as part of the RNA interference response: short-interfering (si)RNAs and PIWI-interacting (pi)RNAs. The siRNA pathway is generally thought to be the main antiviral pathway. Although an antiviral activity has been suggested for the piRNA pathway its role in host defences is not clear. Knock down of key proteins of the piRNA pathway (Ago3 and Piwi5) in Aedes aegypti-derived cells reduced the production of alphavirus chikungunya virus (CHIKV)-specific piRNAs but had no effect on virus replication. In contrast, knock down of the siRNA pathway key protein Ago2 resulted in an increase in virus replication. Similar results were obtained when expression of Piwi4 was silenced. Knock down of the helicase Spindle-E (SpnE), an essential co-factor of the piRNA pathway in Drosophila melanogaster, resulted in increased virus replication indicating that SpnE acts as an antiviral against alphaviruses such as CHIKV and the related Semliki Forest virus (SFV). Surprisingly, this effect was found to be independent of the siRNA and piRNA pathways in Ae. aegypti cells and specific for alphaviruses. This suggests a small RNA-independent antiviral function for this protein in mosquitoes

SpoT Induces Intracellular Salmonella Virulence Programs in the Phagosome.

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures

Conserved themes in small-RNA-mediated transposon control

Eukaryotes are engaged in a constant struggle against transposable elements, which have invaded and profoundly shaped their genomes. Over the past decade, a growing body of evidence has pointed to a role for small RNAs in transposon defense. Although the strategies used in different organisms vary in their details, they have strikingly similar general properties. Basically, all mechanisms consist of three components. First, transposon detection prompts the production of small RNAs, which are Piwi-interacting RNAs in some organisms and small interfering RNAs in others. Second, the population of small RNAs targeting active transposons is amplified through an RNA-dependent RNA polymerase-based or Slicer-based mechanism. Third, small RNAs are incorporated into Argonaute- or Piwi-containing effector complexes, which target transposon transcripts for post-transcriptional silencing and/or target transposon DNA for repressive chromatin modification and DNA methylation. These properties produce robust systems that limit the catastrophic consequences of transposon mobilization, which can result in the accumulation of deleterious mutations, changes in gene expression patterns, and conditions such as gonadal hypotrophy and sterility

Non-coding RNAs in Transcriptional Regulation

Transcriptional gene silencing guided by small RNAs is a process conserved from protozoa to mammals. Small RNAs loaded into Argonaute family proteins direct repressive histone modifications or DNA cytosine methylation to homologous regions of the genome. Small RNA-mediated transcriptional silencing is required for many biological processes, including repression of transposable elements, maintaining the genome stability/integrity, and epigenetic inheritance of gene expression. Here, we will summarize the current knowledge about small RNA biogenesis and mechanisms of transcriptional regulation in plants, Drosophila, Caenorhabditis elegans, and mice. Furthermore, a rapidly growing number of long non-coding RNAs (lncRNAs) have been implicated as important players in transcription regulation. We will discuss current models for long non-coding RNA-mediated gene regulation

Employing transposon mutagenesis to investigate foot-and-mouth disease virus replication

Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part to the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for viral replication as well as locations that can tolerate small genetic insertions. Such insertion sites can be subsequently adapted by the incorporation of commonly used epitope tags and so facilitate their detection with commercial available reagents. In this study, we use random transposon-mediated mutagenesis to produce a library of 15 nucleotide insertions in the FMDV nonstructural polyprotein. Using a replicon-based assay we isolated multiple replication-competent as well as replication-defective insertions. We have adapted the replication competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins, for the use in a variety of downstream assays. Additionally, we show that replication of some of the replication-defective insertion mutants can be rescued by co-transfection of a 'helper' replicon, demonstrating a novel use of random mutagenesis to identify inter-genomic trans-complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornaviral replication complexes

Ribosome Biogenesis Modulates Ty1 Copy Number Control in Saccharomyces cerevisiae.

Transposons can impact the host genome by altering gene expression and participating in chromosome rearrangements. Therefore, organisms evolved different ways to minimize the level of transposition. In Saccharomyces cerevisiae and its close relative S. paradoxus, Ty1 copy number control (CNC) is mediated by the self-encoded restriction factor p22, which is derived from the GAG capsid gene and inhibits virus-like particle (VLP) assembly and function. Based on secondary screens of Ty1 cofactors, we identified LOC1, a RNA localization/ribosome biogenesis gene that affects Ty1 mobility predominantly in strains harboring Ty1 elements. Ribosomal protein mutants rps0bΔ and rpl7aΔ displayed similar CNC-specific phenotypes as loc1Δ, suggesting that ribosome biogenesis is critical for CNC. The level of Ty1 mRNA and Ty1 internal (Ty1i) transcripts encoding p22 was altered in these mutants, and displayed a trend where the level of Ty1i RNA increased relative to full-length Ty1 mRNA. The level of p22 increased in these mutants, and the half-life of p22 also increased in a loc1Δ mutant. Transcriptomic analyses revealed small changes in the level of Ty1 transcripts or efficiency of translation initiation in a loc1Δ mutant. Importantly, a loc1Δ mutant had defects in assembly of Gag complexes and packaging Ty1 RNA. Our results indicate that defective ribosome biogenesis enhances CNC by increasing the level of p22, and raise the possibility for versatile links between VLP assembly, its cytoplasmic environment, and a novel stress response

Interaction Between The MEC1-Dependent DNA Synthesis Checkpoint And G1 Cyclin Function In Saccharomyces Cerevisiae

The completion of DNA synthesis in yeast is monitored by a checkpoint that requires MEC1 and RAD53. Here we show that deletion of the Saccharomyces cerevisiae G1 cyclins CLN1 and CLN2 suppressed the essential requirement for MEC1 function. Wild-type levels of CLN1 and CLN2, or overexpression of CLN1, CLN2, or CLB5, but not CLN3, killed mec1 strains. We identified RNR1, which encodes a subunit of ribonucleotide reductase, as a high-copy suppressor of the lethality of mec1 GAL1-CLN1. Northern analysis demonstrated that RNR1 expression is reduced by CLN1 or CLN2 overexpression. Because limiting RNR1 expression would be expected to decrease dNTP pools, CLN1 and CLN2 may cause lethality in mec1 strains by causing initiation of DNA replication with inadequate dNTPs. In contrast to mec1 mutants, MEC1 strains with low dNTPs would be able to delay S phase and thereby remain viable. We propose that the essential function for MEC1 may be the same as its checkpoint function during hydroxyurea treatment, namely, to slow S phase when nucleotides are limiting. In a cln1 cln2 background, a prolonged period of expression of genes turned on at the G1-S border, such as RNR1, has been observed. Thus deletion of CLN1 and CLN2 could function similarly to overexpression of RNR1 in suppressing mec1 lethality

The Trk Potassium Transporter Is Required for RsmB-Mediated Activation of Virulence in the Phytopathogen Pectobacterium wasabiae

Pectobacterium wasabiae (previously known as Erwinia carotovora) is an important plant pathogen that regulates the production of plant cell wall-degrading enzymes through an N-acyl homoserine lactone-based quorum sensing system and through the GacS/GacA two-component system (also known as ExpS/ExpA). At high cell density, activation of GacS/GacA induces the expression of RsmB, a noncoding RNA that is essential for the activation of virulence in this bacterium. A genetic screen to identify regulators of RsmB revealed that mutants defective in components of a putative Trk potassium transporter (trkH and trkA) had decreased rsmB expression. Further analysis of these mutants showed that changes in potassium concentration influenced rsmB expression and consequent tissue damage in potato tubers and that this regulation required an intact Trk system. Regulation of rsmB expression by potassium via the Trk system occurred even in the absence of the GacS/GacA system, demonstrating that these systems act independently and are both required for full activation of RsmB and for the downstream induction of virulence in potato infection assays. Overall, our results identified potassium as an essential environmental factor regulating the Rsm system, and the consequent induction of virulence, in the plant pathogen P. wasabiae.info:eu-repo/semantics/publishedVersio